1. Field of the Invention
The present invention relates to an immunomodulatory protein and its production method, and more particularly to an immunomodulatory protein cloned from Ganoderma microsporum with immunomodulator efficiency that is better than the immunomodulatory protein isolated from Ganoderma lucidum. 
2. Description of the Related Art
Ganoderma or lingzhi, a natural herbal medicinal fungus that has been used in China since 100 AD, is belonged to Kingdom Fungi, Phylum Basidiomycota, Class Hymenomycetes, Order Aphyllophorales, Family Ganodermataceae, and Genus Ganoderma. There are around 300 different species of Ganoderma, but only a limited number of strains such as Ganoderma lucidum, G. tsugae, G. capense, G. boninense, G. resinaceum, G. sinense, G. japonicum, and G. applanatum were investigate and utilized in the field of pharmacology and clinical studies. After decades of pharmacology studies, Ganoderma or lingzhi was found to contain the active ingredients within its extracts that have the calming, analgesia, heart protecting, liver supporting, blood pressure lowering, lipid lowering cholesterol lowering, anti-allergic, anti-inflammation, anti-virus, anti-tumor and immunomodulation effects.
In 1971, Sasaki et al. found that the polysaccharides of G. applanatu existed the anti-tumor activity and the polysaccharides had become the first proven active ingredient in Ganoderma or lingzhi. The acting mechanism of polysaccharides was not to directly kill or suppress cancer cells but to activate the T cells and increased the ability of natural killer cells to improve immunity, and consequently indirectly express its anticancer activity. In addition, it enhanced the devouring ability of monocyte macrophage and promotes cell endocrine that was able to suppress tumor growth, such as the synthesis and release of interleukin (IL-2<L-4), interferon (IFN-γ and tumor necrosis factor (TNF-α).
Another active ingredient of Ganoderma or lingzhi, immunomodulatory protein, was isolated from the mycelium of G. lucidum by the Japanese research group and named as LZ-8 (Ling Zhi-8). LZ-8, consisting of 110 amino acids with molecular weight of 12,420 Da, were similar to the amino acid sequence and the secondary structure of the immunoglobulin heavy chain variable region. The native LZ-8, existing in the form of homodimer, has the effects of facilitating lymphocytes multiplication and suppressing systemic anaphylaxis reaction and Arthus reaction. Since there was no agglutination/hemagglutination reaction toward human red blood cells, LZ-8 has demonstrated the potential in the human medical applications. The related patents of LZ-8 include the patents of JP2032026, JP3172184 and JP5068561 that use LZ-8 as anti-HIV drug and its nucleotide sequence, as well as the patents of EP0288959B1 and U.S. Pat. No. 5,334,704 that are consisted of the LZ-8 protein characterization and used as immunosuppressive drugs. Two partial sequences of the glycoprotein of LZ-8, -Leu-Ala-Trp-Asp-Val-Lys-(SEQ ID NO:24) and -Asn-Leu-Gly-Val-Lys-Pro-Ser-Tyr-Ala-Val-(SEQ ID NO:25), were also protected by patent (U.S. Pat. No. 5,334,704).
LZ-8, like lectin, has the abilities of agglutinating cells and promoting lymphocyte multiplication. Lectin has the carbohydrate specific binding ability, and such carbohydrate specific binding characteristics allows it to bind with specific carbohydrate on cell surface to stimulate cells and to trigger the follow-up immune responses. Kino et al. discovered that LZ-8 was able to stimulate the multiplication of muridae spleen cells and prevented the Arthus reaction and systemic anaphylaxis reaction in 1989. The further research indicated that LZ-8 was able to effectively suppress the occurrences of autoimmune type-I diabetes in nonobese diabetic (NOD) mice. Furthermore, LZ-8 was able to significantly delay the repulsion time after the pancreas transplantation, comparing to other immunomodulatory drugs, CsA (cyclosporin A, a peptides derived from fungi with immunosuppressing effect) and FK506 (tacrolimus, an antibiotics secreted by soil-borne fungi with immunosuppressing effect).
Both CsA and FK506 existed potential poisoning toward pancreas, while no such poisoning was found toward pancreas for LZ-8. LZ-8, whether the experiments were done in vitro or in vivo, showed immunomodulatory activity, but the detailed mechanism was not clear. In 1991, Kino et al. found that LZ-8 was able to suppress the production of mouse antibodies and speculated that LZ-8 was able to suppress the systemic anaphylaxis reaction and Arthus reaction through blocking off the antibody production. Later, LZ-8 was found to be able to adjust the interactions among cells through controlling the molecules attached onto cell surfaces, and such interaction was not found in autoimmune disease patients.
Ganoderma lucidum immunomodulatory protein, LZ-8, has shown that even simple peptides has the immunomodulatory effect. Other researches also found that the immunomodulatory protein, FIP-gts (fungal immunomodulatory protein-gts), purified from the mycelium of G. tsugae, with molecular weight around 13 kD has the same amino acid sequence as LZ-8. FIP-gts was able to promote the proliferation of human peripheral lymphocytes and mouse spleen cells. In addition, it was able to purify immunomodulatory proteins with molecular weight around 13 kD from non-Ganoderma or lingzhi fungi, for example, FIP-fve from Flammulina velutipes and FIP-vvo from Volvariella volvacea. LZ-8, FIP-fve and FIP-vvo are similar to the variable region of immunoglobulin heavy chains. FIP-fve and FIP-vvo are similar to LZ-8 not only in the sequence and structure but also in the physiological activity.
The healthcare effects of medicinal fungi are well known and their active ingredients were purified or isolated bit by bit. Most of currently known effective ingredients are glucans or pentacyclic tritterpenoid compounds. However, these active ingredients cannot eliminate the interferences of peptidoglycan and proteoglycan during purification. Besides, these active ingredients are either the constituents of cell walls or the secondary metabolites and are difficult in mass production through hetero-expression system.